Engineering an Artificial Membrane Vesicle Trafficking System

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Engineering an artificial membrane vesicle trafficking system (AMVTS) for the excretion of #-carotene in Escherichia coli Tao Wu, Siwei Li, Lijun Ye, Dongdong Zhao, Feiyu Fan, Qinyan Li, Bolin Zhang, Changhao Bi, and Xueli Zhang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00472 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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ACS Synthetic Biology

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Engineering an artificial membrane vesicle trafficking system

2

(AMVTS) for the excretion of β-carotene in Escherichia coli

3

Tao Wua,b+, Siwei Lib+, Lijun Yeb, Dongdong Zhaob, Feiyu Fanb, Qinyan Lib, Bolin Zhangc,

4

Changhao Bi*b, Xueli Zhang*b

5

aCollege

6

bTianjin

Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P R China.

7

cCollege

of Biological Sciences and Technology, Beijing Forestry University, Beijing100083, PR China.

8

*To

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Tao Wu and Siwei Li have contributed equally to this work.

of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300314, P R China

whom correspondence should be addressed: C.B. (email: [email protected]) or X.Z. (email: [email protected])

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ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by

27

natural transportation systems. These products accumulate inside the cells and affect normal

28

cellular physiological functions, which hinders further improvement of carotenoid production by

29

microbial cell factories. In this study, we proposed to construct a novel artificial transport system

30

utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow

31

the physiological mechanism of membrane dispersion to be reconstructed and amplified to

32

establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few

33

proteins in E. coli were reported or proposed to be related to the formation mechanism of outer

34

membrane vesicles, and were individually knocked out or overexpressed to test their physiological

35

functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI

36

resulted in a 13.7% increase of secreted β-carotene with a 35.6% increase of specific production.

37

To supplement the loss of membrane components of the cells due to the increased membrane

38

vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While

39

overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted

40

β-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold

41

increase of secreted β-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time,

42

the specific production of β-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase.

43

The AMVTS was also applied to a β-carotene hyperproducing strain, CAR025, which led to a

44

24-fold increase of secreted β-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the

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specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built

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in this study establishes a novel artificial transport mechanism different from natural protein-based

47

cellular transport systems, which has great potential to be applied to various cell factories for the

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excretion of a wide range of hydrophobic compounds.

49 50

Keywords: AMVTS, outer membrane vesicles, β-carotene, production,

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Escherichia coli

52 53 54 55 56 57 2


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58

Introduction

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Carotenoids are some of the most valuable and abundant natural products. One of the most

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prominent carotenoids, β-Carotene, has been widely used in the pharmaceutical, nutraceutical,

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cosmetics and food industries1, 2. With the development of metabolic engineering, many important

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carotenoids have been successfully produced in engineered microorganisms3-11. In particular,

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Escherichia coli has been extensively engineered for β-carotene production12-14.

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Many studies have focused on increasing precursor supply. For example, the native

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2-C-methyl-D-erythritol-4-phosphate (MEP) pathway was modulated to supply isopentenyl

66

pyrophosphate (IPP), which is the precursor of carotenoids15,

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(including TCA and PPP) were engineered to increase the supply of pyruvate and

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glyceraldehyde-3-phosphate, two important precursors for the MEP pathway17. In addition, a

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heterologous mevalonate (MVA) pathway18,

70

production, and a type IIs restriction-based combined modulation technique was established to

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optimize the MVA pathway20. The cellular pools of the cofactors ATP and NADPH were also

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enhanced to improve β-carotene production17. Recently, new cellular modules were engineered. In

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one new module, the membrane was engineered to provide more space for β-carotene storage and

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hence production21.

19

16.

Central metabolic modules

was introduced into E. coli to increase IPP

75

Carotenoids are large, hydrophobic molecules. They accumulate inside cells, and cannot be

76

effectively excreted, which might inhibit the corresponding synthesis pathways, affect normal

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cellular physiological functions, and hinder further improvement of carotenoid cell factories21, 22.

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In addition, it is time consuming and costly to extract β-carotene from microbial cell factories23.

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However, most studies have focused on engineering metabolic pathways, and minimal research

80

has been conducted on transporting the carotenoid products outside of the cell. Only one article

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described an attempt to employ the ABC transporter MsbA for exporting carotenoid compounds,

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but this transporter achieved a very low efficiency24. It was suggested that large hydrophobic

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molecules, such as carotenoids, cannot be effectively excreted by natural transport systems.

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Therefore, it is necessary to create a novel artificial transport system that does not rely on natural

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transport proteins and protein channels that can efficiently transport large hydrophobic molecules.

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Since carotenoids mainly accumulate within the cell membrane compartment21, membrane

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components might be used as carriers to excrete these compounds. The secretion of extracellular

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vesicles is universal from bacteria to humans and plants25,

89

components including nucleic acids, lipids, and proteins between cells. They can act as signaling

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vehicles in normal cellular homeostatic processes or can be released as a consequence of

91

pathological developments27, 28. Gram-negative bacteria were reported to weakly shed membrane

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components in some environments in the form of outer membrane vesicles (OMVs), which are

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nanoscale proteoliposomes with some physiological roles28-31. OMVs can neutralize

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environmental agents that target the outer membrane32, aid in the release of attacking phages33,

95

remove misfolded periplasmic proteins34, and nucleate the formation of bacterial communities35.

26,

3


and allows for the exchange of


96

A few proteins in E. coli were reported or proposed to be related to the formation of outer

97

membrane vesicles, which we might be able to manipulate to enhance the OMV system. For

98

example, the Tol-Pal complex, a cell-division component that aids in the invagination of the outer

99

membrane and in the stability of the inner membrane, is composed of the inner membrane proteins

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TolA, TolQ, and TolR associated via their trans-membrane segments36, 37. The TolB periplasmic

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protein interacts with the Pal outer membrane lipoprotein. A mutation in any of these proteins

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might lead to a defect in outer membrane integrity and a concomitant increase in the production of

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OMVs38. In addition, envelope stability relies on envelope crosslinks, including the covalent

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crosslinking of lipoprotein (Lpp) in the outer membrane with the peptidoglycan (PG) saccules39,

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and the non-covalent interactions between the PG and outer membrane protein A (OmpA). To

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supplement the possible loss of membrane components from cells by increased dispersion of

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membrane vesicles, the synthesis pathway of phosphatidyl ethanolamine needs to be enhanced.

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Furthermore, based on information from chapter 37 of the classic book Escherichia coli and

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Salmonella,40 membrane lipids are all synthesized in the cytoplasm or near the inner membrane,

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and some of these are then transferred to the outer membrane. Therefore, the lipophilic β-carotene

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might be transported via such a lipid flux. In addition, the inner and outer membranes are normally

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not connected, but they nevertheless tend to form contacts sometimes and some membrane

113

components can be exchanged. These are probably the mechanisms by which β-carotene is

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deposited in the inner membrane to be translocated to the OMVs via the periplasm and outer

115

membrane. Thus, in this study, we reconstructed the natural OMV system to establish a novel

116

artificial membrane vesicle transport system (AMVTS) and introduce it into E. coli cell factories

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for efficient β-carotene excretion.

118 119

Results and Discussion

120

Deleting OMV-related genes enabled β-carotene excretion

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A few proteins in E. coli were reported to be related to the mechanism of membrane vesicle

122

formation, based on which we projected several more candidates from reported protein

123

information. To study the function of these proteins related to the formation of outer membrane

124

vehicles (OMVs) and the possible OMV-mediated β-carotene excretion, the genes encoding the

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candidate proteins were knocked out or overexpressed in the β-carotene producing strain CAR015

126

(Table 1). While overexpression of the candidate genes fliC (a flagellar filament structural

127

protein), nlpA (a nonessential periplasmic lipoprotein tethered to the inner membrane), and pepP

128

(proline aminopeptidase) did not improve β-carotene excretion (Figure. 1B), knocking out tolA,

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tolR, nlpI, nlpD (lipoprotein that is the activator of AmiC murein hydrolase activity), ompF (outer

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membrane porin), and pnP (polynucleotide phosphorylase/polyadenylase) did increase the

131

excreted β-carotene content41 (Figure 1A). The strain TW-002 with tolA knocked out had the

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highest secreted β-carotene content of 0.62 mg/g DCW, which was followed by strains TW-003

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and TW-004, with tolR and nlpI knocked out. respectively. The results showed that knocking out 4


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tolA, tolR and nlpI individually enhanced the secreted β-carotene content compared with the

135

parent strain CAR015, which only secreted 0.15 mg β-carotene /g DCW .

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The only known physiological function of TolA is in the Tol-Pal complex where it aids in

137

outer membrane and inner membrane stability and is related to OMVs. It is quite possible that this

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modification enhanced the OMVs formation mechanism and increased OMV-mediated β-carotene

139

excretion. In addition, higher specific production of β-carotene was obtained with the TW-002

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strain. The results might support our hypothesis that when the cells accumulate the large

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hydrophobic molecules, carotenoids are removed from the cells, the inhibition of synthesis

142

pathway is removed, and normal cellular physiological functions are restored, which is beneficial

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for the carotenoid cell factories.

144 145 146

Further increase of β-carotene excretion by the combined deletion of tolA, tolR and nlpI genes

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Since knocking out genes tolA, tolR or nlpI individually was found to increase β-carotene

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excretion, a combined knockout strategy was employed to identify the best combination for

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further improvement of β-carotene production. The corresponding multiple-knockout strains

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△tolA△tolR, △tolA△nlpI or △tolR△nlpI were constructed based on strain CAR015 to obtain

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strains TW-011 to TW-013, respectively, which were subjected to production analysis. As

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illustrated in Figure 2, all strains with combined gene deletions had increased production of

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excreted β-carotene, among which, TW-012 with both tolA and nlpI knocked out had the highest

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excreted β-carotene content, at 2.21 mg/g DCW.

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Both TolA and TolR belong to the Tol-Pal complex, which is a transmembrane multiprotein

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complex that forms bridges between the outer membrane, peptidoglycan, and the inner membrane,

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and is generally required for outer membrane integrity42, 43. NlpI is related to protein expression

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and localization of the outer membrane34,

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membrane protein localization and the Tol-Pal system had a synergistic effect on OMV formation,

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which further enhanced the production of outer membrane vesicles. The established AMVTS not

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only increased secreted β-carotene, but also increased its total specific production.

44.

The results indicated that disruption of outer

162 163

Modulating membrane synthesis pathways facilitated AMVTS-mediated β-carotene

164

excretion

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Theoretically, the enhanced OMV system might disperse large amounts of components out of

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the integrated cell membrane. Thus, increased synthesis of membrane components might

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complement the loss of cell membrane material and be beneficial for the AMVTS. The major

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component of the E. coli membrane is phosphatidyl ethanolamine (PE), which accounts for about

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70-80% of lipid content of the membrane, which also contains 20% phosphatidylglycerols and

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5-10% cardiolipin45. The synthesis pathway of phosphatidyl ethanolamine is illustrated in Figure

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3. In this pathway, the intermediate diacylglycerol-3-P also serves as a precursor for other

172

membrane

components46.

To

study

how

the

membrane

5


synthesis

pathways

affect


173

AMVTS-mediated β-carotene excretion, they were divided into four modules, which were

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overexpressed in strains with AMVTS to analyze their effect on β-carotene excretion (Figure 3).

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To understand the general trend of the impact of the membrane synthesis pathways, six strains

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(TW002, 003, 004, 011, 012, 013) were selected for analysis. As illustrated in Figure 4, module I

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expressed from plasmid pAcc had the highest impact on AMVTS-mediated β-carotene excretion.

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While the non-AMVTS parent strain still had low excretion, TW-002, TW-003, TW-012 and

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TW013 all increased their β-carotene excretion greatly. In particular, TW-012 and TW013 had

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4.43 and 4.72 mg/g DCW of secreted β-carotene, which accounted for one third of the total

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β-carotene production (Figure 4A). In addition, module II expressed from plasmid pFAS and

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module III expressed from plasmid pPlsBC were found to have the general trend of promoting

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β-carotene excretion of the tested strains to some extent.

184

As shown in Figures 3 and 4(A), enhanced Acc expression provided more acyl-ACP, which

185

is the core precursor for the synthesis of all membrane components. FAS has a similar function.

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The specific production of secreted β-carotene by strain TW013 (pAcc) was 4.72 mg/g DCW,

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which was a 16-fold increase compared with the parent strain CAR015 (pBad-M), and about a

188

4-fold increase compared with the starting strain TW-013. The specific production of secreted

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β-carotene in strain TW013 (pFAS) was 2.28 mg/g DCW, which was a 15-fold increase compared

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to CAR015 (pTrc99A-M), and a 1.5-fold increase compared to strain TW-013. Overexpression of

191

plsB and plsC produced a sufficient amount of diacylglycerol-3-P, which is an important precursor

192

for the synthesis of both phosphatidyl ethanolamine and cardiolipin. As a result, the number of

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total membrane components was increased. When the module was introduced into strains TW-012

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and TW-013, the membrane components lost due to AMVTS were replaced, and the amount of

195

excreted β-carotene in both strains was increased.

196

However, we found that the PE synthetic module carried on pPE had only a slight effect on

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the specific production of β-carotene. This was probably because the formation of PE reduced the

198

pool of the universal precursor diacylglycerol-3-P, and the balance of the membrane components

199

was changed to a status not optimal for the AMVTS.

200 201

Further increase of β-carotene excretion by combined overexpression of membrane

202

synthesis genes

203

Since modules I, II and III were found to be beneficial for AMVTS-mediated β-carotene

204

excretion, the combined expression of these modules was studied to obtain the optimal expression

205

pattern for AMVTS. Combinations of Acc and FAS modules, Acc and PlsBC modules, FAS and

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PlsBC modules, and all three modules were overexpressed in the best-performing strains, TW-012

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and TW-013. As illustrated in Figure 5, all expression combinations increased the amount of

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secreted β-carotene, and we found that combined expression of the Acc and PlsBC modules

209

yielded the best results. While both strains had high β-carotene secretion levels, TW-013 (pAcc,

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pPlsBC) was better, with 10.72 mg/g DCW β-carotene secreted to the dodecane layer, which was 6


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10.5-fold higher than that of the starting strain TW-013 and accounted for almost half of the total

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β-carotene production. Surprisingly, the total specific production of β-carotene also increased to

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21.88 mg/g DCW, which was a 94% increase compared to the starting strain TW-013.

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Acyl-ACP and diacylglycerol-3-P are two important precursors in the synthesis pathway of

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membrane components. Overexpression of both Acc and FAS was not better than overexpression

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of Acc alone, while co-expression of Acc and PlsBC led to much higher production than the

217

overexpression of Acc. This result indicated that with overexpression of Acc, enough acyl-CoA

218

was present so that FAS was not rate-limiting, while the conversion of acyl-CoA to

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diacylglycerol-3-P was. Combined overexpression of all three modules did not produce more than

220

the combination of Acc and PlsBC, which indicated that when Acc and PlsBC were both

221

expressed, FAS was at a suitable strength. Further overexpression of FAS might affect this

222

balance. When modules I and III were overexpressed simultaneously, the entire pathway was

223

enhanced, and the number of total membrane structures was increased, which explained the

224

synergistic effect of these two modules in the AMVTS strains TW-012 and TW-013.

225 226

Microscopic observation of E. coli cells that excrete β-carotene via AMVTS

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To directly observe and study the morphology of E. coli cells that excrete β-carotene via

228

AMVTS, electron microscopy was performed on the best stain TW-013(pAcc, pPlsBC) (Figure 6).

229

Cells of the parent strain CAR015 had a normal rod shape with integrated and smooth cell

230

membranes, and there were no vesicles outside the membrane, which showed that the β-carotene

231

producers had normal cellular and membrane morphology, and suggested that β-carotene

232

production itself did not affect the cell membrane. On the other hand, significant morphological

233

differences were observed in the AMVTS strain TW-013(pAcc, pPlsBC), with possible membrane

234

vesicles around the outer membrane that were similar to previously reported observations 47-49 . In

235

addition, while the cells kept the rod shape, TW-013(pAcc, pPlsBC) possibly had a less integrated

236

membrane than its parent strain. Thus, the microscopic observations directly illustrated that an

237

artificial membrane vesicles system for the β-carotene microbial cell factory was indeed

238

successfully established using our as proposed engineering strategy.

239 240

The secreted β-carotene was mainly associated with the excreted OMVs

241

In order to demonstrate that the secreted β-carotene was mainly stored in OMVs of E. coli

242

cells, we extracted the OMVs from both the control and engineered strains using a classic OMV

243

extraction method

244

the control strain, different amounts of OMVs were obtained from the engineered OMV excretion

245

strains. Furthermore, based on the orange color of the OMV pellets, β-carotene could be visibly

246

observed. The figure qualitatively suggested that β-carotene was secreted with the OMVs, and the

247

TW013(pAcc, pPlsBC) strain had the most secreted β-carotene. As is shown in figure S1, the

248

peaks of β-carotene from CAR015 was small and clean, and there was a very small tail in the β-carotene peak from TW002. The HPLC sample was acetone extracted

249

50, 51.

As can be seen from in figure 7, while little OMVs were obtained from

7



250

β-carotene from to the dodecane layer and diluted to a concentration for injection.

251

The β-carotene contents of the OMVs from these strains was also analyzed. As shown in

252

Figure 7, all tested strains sequestered β-carotene in the OMVs. Especially strain TW-013(pAcc,

253

pPlsBC) had 4.5 mg/g DCW β-carotene secreted with the OMVs, while the control strain had only

254

0.05 mg/g DCW. Although the extracted β-carotene content from the OMVs was lower than the

255

10.7 mg/g DCW obtained with the addition of n-dodecane, considering the complex cell

256

manipulation procedure and β-carotene extraction procedure, we considered the loss reasonable.

257

To determine whether the excretion of β-carotene was due to cell lysis, the growth status of

258

the engineered strains was measured via the OD600 as shown in Table S2. The OD values of some

259

of the engineered strains were lower than that of the control strain, but not significantly. For

260

example, the OD value of strain TW-013(pAcc, pPlsBC) decreased 28% compared to the control

261

strain CAR015, but its secreted β-carotene was 53-fold that of CAR015 (increasing from 0.2 to

262

10.7 mg/g DCW). Moreover, the specific production value of β-carotene increased from 6.9 to

263

21.9 mg/g DCW, a 3.2-fold increase. The extent of the OD value decrease was lower than the

264

increase of the specific production and secretion of β-carotene, indicating that our engineering

265

strategy was efficient. Since the introduction of plasmids and expression of heterogenous protein

266

is known to affect cell growth,52, 53 we considered the mild growth defect of the engineered strains

267

as a normal growth burden effect. Secondly, the viable count of strain TW-013(pAcc, pPlsBC)

268

was slightly lower than that of the control strain CAR015 (TableS3), which was in agreement with

269

the OD values of both strains, suggesting that there was no excessive cell death in the culture of

270

TW-013(pAcc, pPlsBC).

271

Additionally, protein mass spectrometry was used to analyze the proteins present in the

272

culture broth outside the cells. The results of the control and hyperproducing strains are shown in

273

Tables S4 and S5. The emPAI value is used for determining the relative protein quantity, while the

274

Sum PEP Score, Score Sequest HT and PSMs values are used to detect the protein amount

275

indirectly. It could be seen that after the fermentation process, the culture supernatants of both

276

control and engineered cells contained intracellular proteins, such as RpmC, RpmE and RpmG,

277

which suggested that both cultures contained lysed cells. In addition, based on the emPAI and

278

PSMs value, TW-013(pAcc,pPlsC) and CAR015 had similar levels of emPAI and PSMs in protein

279

RpmG; and the emPAI and PSMs values of protein RpmE and RpmC in TW-013(pAcc,pPlsBC)

280

were lower than that of CAR015, These results indicating that modification of the outer membrane

281

vesicles mildly affected cell growth, but did not result in significantly increased cell lysis

282

compared with the control strain. Thus, the excretion of β-carotene was not due to the lysis of cells,

283

but due to the excretion of OMVs.

284 285

AMVTS improved β-carotene production in the hyperproducer strain

286

To further demonstrate the capacity of AMVTS, it was introduced into the β-carotene

287

hyperproducing strain CAR025. TolA and nlpI were knocked out to obtain TW-015, and tolR and

288

nlpI were knocked out to obtain TW-016. The synthesis pathway genes of membrane components 8


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289

were overexpressed in both strains. Both strains were found to have improved β-carotene

290

excretion, while one strain further benefited from the excretion and had increased β-carotene

291

production. In particular, 12.7 mg/g DCW β-carotene was excreted by TW-015 (pAcc, pPlsBC),

292

and its total specific production of β-carotene was increased to 44.8 mg/g DCW, which was

293

24-fold and 60% higher than the respective values of the parent strain (Figure 8).

294

Recently, lipid engineering and systematic metabolic engineering were combined in

295

Saccharomyces cerevisiae for high-yield production of lycopene. In fed-batch fermentation, a

296

lycopene production of 2.37 g/L and 73.3 mg/g CDW was reached, representing the highest

297

lycopene yield in Saccharomyces cerevisiae to date54. Since S. cerevisiae can produce

298

extracellular vesicles55, which function similarly to OMVs in some aspects, this strategy might be

299

applied to eukaryotes in the future.

300 301

Conclusions

302

In this study, a novel transport system, termed AMVTS, was built to excrete β-carotene from

303

E. coli cells independent of membrane proteins. With this engineering strategy, a superior

304

β-carotene excreting strain was obtained, which could secrete 10.72 mg/g DCW β-carotene out of

305

the cells, a 71.5-fold increase compared with the parent strain CAR015 (Figure 9). The total

306

specific production value also increased to 21.88 mg/g DCW, a 3.2-fold increase compared to

307

CAR015. When AMVTS was introduced into the hyperproducing strain CAR025, 12.7 mg/g

308

DCW β-carotene was excreted and a higher specific production of 44.8 mg/g DCW was achieved,

309

which was 24-fold and 60% higher than the respective values of the parent strain. The results

310

support our hypothesis that carotenoid cell factories benefit from the lipid-mediated removal of

311

large hydrophobic molecules that are produced and accumulate in the cells.

312

In summary, AMVTS represents a novel strategy for establishing an excretion system for

313

large hydrophobic molecules, which cannot be transported by protein-based natural systems. This

314

engineering strategy might improve the excretion and production of a wide spectrum of

315

hydrophobic products. It is quite possible that in addition to the bacterial cell factories we studied,

316

eukaryotic cell factories, plants and even animal producers might benefit from such a novel

317

lipid-carrier-based transportation system specific for hydrophobic molecules.

318 319

Materials and Methods

320

Strains, media and culture conditions

321

The bacterial strains used for DNA manipulation and β-carotene production in this study are

322

listed in Table 1. The β-carotene-producing strains CAR015 and CAR025 were used as the parent

323

strains for AMVTS engineering. For strain construction, cultures were grown aerobically at 30 or

324

37C in Luria-Bertani medium (per liter: 10 g tryptone, 5 g yeast extract and 10 g NaCl). For

325

β-carotene production, single colonies were picked from LB plates and transferred into 15 mm × 9



326

100 mm tubes containing 4 ml of LB with or without 34 mg/L of chloramphenicol and cultured at

327

37C and 250 rpm overnight. The resulting seed cultures were used to inoculate 100 ml flasks

328

containing 10 ml of fermentation medium (LB containing 2% glycerol with or without

329

chloramphenicol ampicillin and kanamycin) to an initial OD600 of 0.05 and grown at 30 °C and

330

250 rpm. For strains bearing the Ptrc promoter, 0.1 mM IPTG was added 3 h after inoculation,

331

which was followed by 45 h of induced expression. After 48 h of growth, the cells were collected

332

to measure the β-carotene production.

333 334

Plasmid construction

335

All plasmids used in this study are listed in Table 1. Plasmids were assembled using the

336

Golden Gate method56. To construct the plasmids expressing the OMV-related genes, fliC, nlpA,

337

and pepP were amplified via PCR using chromosomal DNA of E. coli ATCC 8739 as the

338

template, and cloned into vector pACYC184-M. To construct the plasmids for FAS synthesis, FAS

339

from Corynebacterium glutamicum ATCC 13032 was cloned into pTrc-99A-M. E. coli

340

acetyl-CoA carboxylase genes AccA, B, C and D were amplified via PCR using chromosomal

341

DNA of E. coli ATCC 8739 as the template and cloned into plasmid pBAD-rfp with Ptrc promoter

342

to construct the expression plasmids. The E. coli phosphatidyl ethanolamine synthesis genes plsB,

343

plsC, cdsA, pssA, and psd were amplified via PCR using chromosomal DNA of E. coli ATCC

344

8739 as the template and cloned into plasmid pACYC184-M to construct the expression plasmids.

345

All primers were synthesized by Genewiz (Beijing, China) and are listed in Table S1. Gene

346

sequencing was also carried out by Genewiz.

347 348

Genome editing

349

Genome editing in this work was performed using the CRISPR/Cas9 genome editing protocol

350

as described previously57. The homologous arms of target loci were amplified via PCR using

351

chromosomal DNA of E. coli ATCC 8739 as the template and assembled with the N20PAM

352

fragment using the Golden Gate method. The resulting edited DNA cassettes were introduced into

353

the target strains along with the universal editing plasmid to conduct the editing process.

354 355

Analysis of β-carotene production and secreted β-carotene content

356

The β-carotene titers were quantified by measuring the absorption at 453 nm of acetone

357

extracts of the cells as described previously14, with some modifications as follows: Cells were

358

harvested by centrifugation at 16,200 g for 3 min, suspended in 1 mL of acetone, incubated at

359

55°C for 15 min in the dark and centrifuged at 16,200 g for 10 min. The acetone supernatant

360

containing β-carotene was transferred to a new tube, and the β-carotene content was analyzed at

361

453 nm using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). Dry cell weight

362

(DCW) was calculated according to the empirical formula: 1 OD600 = 0.323 g DCW/L.

363

N-dodecane was chosen for the extraction of hydrophobic carotenoid due to its low toxicity

364

to E. coli58 and high hydrophobicity (log PO/W, 6.6); n-dodecane was added to the culture at a 10


Page 10 of 25

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


365

volume ratio of 1:10 to form a hydrophobic phase above the culture phase. During fermentation,

366

secreted β-carotene was absorbed in the n-dodecane layer. After removing the cells by

367

centrifugation at 8,000 × g for 3 min, the β-carotene content in n-dodecane was obtained and

368

analyzed photometrically at 453 nm using a Shimadzu UV-2550 spectrophotometer (Shimadzu,

369

Kyoto, Japan). The results represent the means ± SD of three independent experiments.

370

Extraction of OMVs from engineered β-carotene-producing E. coli strains

371

Extraction of OMVs was done as described previously50, 51 with some modifications. Briefly,

372

the cultured cells were pelleted at 5,000×g for 10 min. The supernatant was filtered through a 0.45

373

μm pore-size filter to remove the remaining cells and concentrated by ultrafiltration though a

374

100-kDa hollow fiber membrane. OMVs were prepared by pelleting after the centrifugation in a

375

45 Ti rotor (Beckman Instruments, Ireland) at 150,000×g for 2 h at 4°C. Then, the pelleted OMVs

376

were resuspended in phosphate-buffered saline (PBS) and stored at -80°C for further use.

377 378

Proteins extraction and sample preparation

379

To collect proteins in the supernatant for mass spectrometry analysis, the cell protein

380

extraction procedure was as follows: 1) Prepared 50 mL fermentation medium of the E. coli

381

CAR015 and TW-013(pAcc, pPlsBC), then the cells were harvested by centrifugation at 5000× g

382

for 5 min, 2) the cell supernatant was filtered through a 0.22 μm pore-size filter to remove the

383

remaining cells, 3) 7.5 g trichloroacetic acid (TCA) was added to the supernatant and the proteins

384

precipitated at 4°C for 4 h, 4) the protein precipitate was collected by centrifugation at 8000 × g

385

for 5 min, 5) the protein precipitate was washed with acetone three times and then dried, 6) the

386

collected pellet was dissolved in 10 mL protein dissolution buffer (8 M urea, 1 % DTT) and mixed

387

well, then the samples were stored at -80°C for protein mass spectrometry or further analysis. The

388

protein mass spectrometry was performed using the OrbiTrap Fusion LUMOS Tribrid Mass

389

Spectrometer (LC-MS) (Thermo Fisher, USA) as described before59, 60.

390 391

Observation of the cell morphology via transmission electron microscopy

392

Samples were collected at 48 hours after inoculation, washed three times using

393

phosphate-buffered saline (PBS) (pH 7.2), and fixed with 1% glutaraldehyde at 4°C overnight.

394

Cells were resuspended in 1% osmium tetroxide for 5 min at room temperature, centrifuged, and

395

resuspended again in fresh 1% osmium tetroxide for 45 min at room temperature. Then the cells

396

were dehydrated by 15-minute washes in a graded series of ethanol solutions (50%, 70%, 80%,

397

90%, 95%, and 100%). For embedding, cells were incubation for 2h each in 3:1, 1:1, 1:3 mixtures

398

of dehydrating agent/embedding medium, after which the cells were resuspended in pure

399

embedding medium and incubated at room temperature overnight. The next day, samples were

400

resuspended in fresh embedding medium and cured for 24-48 h at 80°C , cut into 60-80 nm

401

sections, stained with uranyl acetate and lead citrate, and observed on a Hitachi HT7700 electron

402

microscope (Hitachi, Japan) operating at 180 kV 61, 62. 11



403 404

Page 12 of 25

Supporting Information Supplementary tables and figures are available in the supporting information

405 406

Author contributions: TW designed and performed the research, analyzed data, and wrote the

407

paper; SL ,LY, DZ, FF designed the research and analyzed data; QL provided the bacteria; CB,

408

XZ designed the research, analyzed data and wrote the paper.

409 410

Competing financial interests: The authors declare no competing financial interests.

411 412

Data and materials availability: The datasets generated during and/or analyzed during the

413

current study are available from the corresponding author on reasonable request.

414 415

Acknowledgements

416

This research was financially supported by the Key Research Program of the Chinese Academy of

417

Sciences (KFZD-SW-215，ZDRW-ZS-2016-3) and the National Natural Science Foundation of

418

China (31522002, 31770105).

419 420

References

421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

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586 587 588

FIGURES LEGENDS

589

Figure 1. Excreted β-carotene and its total specific production value in OMV-engineered E.

590

coli strains

591

(A) β-carotene production values of strains TW-002 to TW-007, with genes tolA, tolR, nlpI, nlpD,

592

ompF or pnP knocked out, respectively. (B) β-carotene production values of strains with genes

593

fliC, nlpA or pepP overexpressed. Three repeats were performed for each strain, and the error bars

594

represent standard deviations.

595 596

Figure 2. Excreted β-carotene and its total specific production values of strains with

597

combined knockout of genes.

598

Combined knockouts of tolA and tolR, tolA and nlpI and tolR and nlpI were performed in strain

599

CAR015 to obtain strains TW-011 to TW-013, respectively. Three repeats were performed for

600

each strain, and the error bars represent standard deviations.

601 602

Figure 3．Synthetic modules for membrane components.

represents multiple reactions.

603 604

Figure 4 ． Excreted β-carotene and its total specific production value of strains with

605

combined overexpression of phosphatidylethanolamine synthesis modules

606

E. coli strains carrying (A) Module I encoded by the plasmid pAcc; (B) Module II encoded by the

607

plasmid pFAS; (C) Module III encoded by the plasmid pPlsBC; (D) Module Ⅳ encoded by the

608

plasmid pPE. Genes involved in the synthesis of membrane components including cdsA, pssA and

609

psd. Three repeats were performed for each strain, and the error bars represent standard

610

deviations.

611 612

Figure 5. Excreted β-carotene and its total specific production value of AMVTS strains with 16


Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


613

combined overexpression of phosphatidyl ethanolamine synthesis modules.

614

(A) Strain TW-012 with combined overexpression of Acc, FAS and PlsBC modules; (B) Strain

615

TW-013 strain with combined overexpression of Acc, FAS and PlsBC modules. Three repeats

616

were performed for each strain, and the error bars represent standard deviations.

617 618

Figure 6．Electron microscopy images of the AMVTS strain TW-013(pAcc, pPlsBC) and

619

the control.

620

Microscopy images include the parent strain CAR015 and AMVTS strain TW-013(pAcc, pPlsBC).

621

White arrows indicate the observed OMV-like vesicles.

622 623

Figure 7. OMVs which contain β-carotene were extracted from engineered β-carotene

624

producing strains.

625 626

Figure 8．The specific production of β-carotene by strains obtained from CAR025 by outer

627

membrane vesiculation enhancement.

628

Overexpression of module I together with module III in strains TW-015 and TW-016. Three

629

repeats were performed for each strain, and the error bars represent standard deviations.

630 631

Figure 9 ． Diagram summarizing the increase of the specific production of β-carotene

632

obtained through the membrane vesicle trafficking system.

633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 17



648 649

Engineering an artificial membrane vesicle trafficking system

650

(AMVTS) for the excretion of β-carotene in Escherichia coli

651

Tao Wua,b+, Siwei Lib+,Lijun Yeb, Dongdong Zhaob, Feiyu Fanb, Qinyan Lib, Bolin Zhangc,

652

Changhao Bi*b, Xueli Zhang*b

653

654 655

TOC

For Table of Contents Use Only

656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 18


Page 18 of 25

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


674 675

Figures and Tables

676 677

Figure 1. Excreted β-carotene and its total specific production value in OMV-engineered E. coli

678

strains

679

(A) β-carotene production values of strains TW-002 to TW-007, with genes tolA, tolR, nlpI, nlpD,

680

ompF or pnP knocked out, respectively. (B) β-carotene production values of strains with genes

681

fliC, nlpA or pepP overexpressed. Three repeats were performed for each strain, and the error bars

682

represent standard deviations.

683 684

Figure 2. Excreted β-carotene and its total specific production values of strains with combined

685

knockout of genes.

686

Combined knockouts of tolA and tolR, tolA and nlpI and tolR and nlpI were performed in strain

687

CAR015 to obtain strains TW-011 to TW-013, respectively. Three repeats were performed for 19



688

Page 20 of 25

each strain, and the error bars represent standard deviations.

689

690 691

Figure 3 Synthetic modules for membrane components.

represents multiple reactions.

692 693 694

20


Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


695 696

Figure 4 ． Excreted β-carotene and its total specific production value of strains with combined

697

overexpression of phosphatidylethanolamine synthesis modules

698

E. coli strains carrying (A) Module I encoded by the plasmid pAcc; (B) Module II encoded by the

699

plasmid pFAS; (C) Module III encoded by the plasmid pPlsBC; (D) Module Ⅳ encoded by the

700

plasmid pPE. Genes involved in the synthesis of membrane components including cdsA, pssA and

701

psd. Three repeats were performed for each strain, and the error bars represent standard

702

deviations.

703 704 705 706 707 21



Page 22 of 25

708 709 A

B

710 711 712 713

Figure 5. Excreted and total β-carotene specific production value of AMVTS strains with

714

combinatory overexpression of phosphatidyl ethanolamine synthesis modules

715

(A) TW-012 strain with combinatory overexpression of Acc, FAS and PlsBC modules; (B)

716

TW-013 strain with combinatory overexpression of Acc, FAS and PlsBC modules. Three repeats

717

were performed for each strain, and the error bars represent standard deviation.

718 719 CAR015

TW-013(pAcc,pPlsBC)

500nm

500nm

720 721 722

Figure 6．Electron microscopy images of the AMVTS strain TW-013(pAcc, pPlsBC) and the

723

control .

724

Microscopy images include the parent strain CAR015 and AMVTS strain TW-013(pAcc, pPlsBC).

725

White arrows indicate the observed OMV-like vesicles. The two tubes show β-carotene containing

726

OMVs in dodecane. 22


Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


727 728

Figure 7. OMVs which contain β-carotene were extracted from engineered β-carotene producing

729

strains.

730 731

732 733

Figure 8 ． The specific production of β-carotene by strains obtained from CAR025 by outer

734

membrane vesiculation enhancement.

735

Overexpression of module I together with module III in strains TW-015 and TW-016. Three

736

repeats were performed for each strain, and the error bars represent standard deviations.

737

23



738 739

Figure 9 ． Diagram summarizing the increase of the specific production of β-carotene obtained

740

through the membrane vesicle trafficking system.

741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 24


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

761


Table 1 Strains and plasmids used in this study Strains and plasmids

Relevant characteristics

Sources

Strains CAR005

ATCC 8739, M1-37::dxs, M1-46::idi

17

M1-93::Crt, M1-46::SucAB, M1-46::sdh, M1-46::talB

CAR015

CAR005, ispG-mRSL-4, ispH-mRSL-14

Lab collection

CAR025

CAR015, replacing the promoter of crtEYIB with Ptrc promoter

Lab collection

TW-002

CAR015, △tolA

This work

TW-003

CAR015, △tolR

This work

TW-004

CAR015, △nlpI

This work

TW-005

CAR015, △nlpD

This work

TW-006

CAR015, △ompF

This work

TW-007

CAR015, △pnP

This work

TW-011

TW-002, △tolR

This work

TW-012

TW-002, △nlpI

This work

TW-013

TW-003, △nlpI

This work

TW-015

CAR025, △nlpI △tolA

This work

TW-016

CAR025, △nlpI, △tolR

This work

Plasmids pACYC184-M

cat; replace tet with lacI and Ptrc of pTrc99A-M

17

bla; PacI, SpeI and NdeI site put in front of the lacI pTrc99A-M

gene, and PacI site put after rrnB T2 transcriptional

17

terminator pBad-M

Replace ara with Ptrc of pBad-ara-M

Lab collection

pFliC

pACYC184-M with Ptrc controlled fliC

This work

pNlpA

pACYC184-M with Ptrc controlled nlpA

This work

pPepP

pACYC184-M with Ptrc controlled pepP

This work

pAcc

pBad-M with Ptrc control AccA, AccB, AccC and AccD

This work

pFAS

pTrc-99A-M with Ptrc controlled Fas

This work

pACYC184-M with Ptrc controlled plsB and plsC

This work

pACYC184-M with Ptrc controlled cdsA.pssA and psd

This work

pPlsBC pPE 762

25


Engineering an Artificial Membrane Vesicle Trafficking System

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